Imaging distal end of multimode fiber

ABSTRACT

Where a multimode fiber is used for light delivery in a microscope system and a transverse distribution of light exiting a distal end of the fiber is substantially uniform, the distal end is imaged onto a plane of a sample to be probed by the microscope system, or at a conjugate plane. Alternatively, the distal end is imaged onto a plane sufficiently close to the sample plane or the conjugate plane such that a radiant intensity of light at the sample plane or the conjugate plane is substantially uniform. In the case of a multi-focal confocal microscope system, the distal end of the multimode fiber is imaged onto a plane of a segmented focusing array. Alternatively the distal end is imaged onto a plane sufficiently close to the segmented focusing array plane such that a radiant intensity of the light at the segmented focusing array plane is substantially uniform.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/267,725, entitled “Imaging LightExiting Multi-Mode Fiber to Achieve Substantially Uniform Illumination”,filed Dec. 8, 2009, and which is incorporated by reference in itsentirety herein. This application also claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/411,434,entitled “Imaging Light Exiting Multi-Mode Fiber to AchieveSubstantially Uniform Illumination”, filed Nov. 8, 2010, and which isincorporated by reference in its entirety herein.

TECHNICAL FIELD

This disclosure is generally related to the technical field of opticalmicroscopy.

BACKGROUND

Optical microscopy involves the projection of light or radiation onto asample, and the subsequent detection of reflected, scattered orfluorescence light from the sample.

One example of an optical microscope system is a traditional pointscanning confocal microscope. In point scanning confocal microscopy, asingle diffraction-limited point of light is projected onto a sample. Byimaging that point onto a single element detector, the reflected,scattered or fluorescence light originating from that point in thesample can be measured. A single pinhole placed at a conjugate imageplane located between the sample and the detector rejects out of focuslight and creates the confocal effect. By scanning the point of light ina manner designed to illuminate the focal plane, for example, by rasterscanning, an image of the sample can be constructed point by point. Bymoving the focal plane optically or by moving the sample, multiple focalplanes can be imaged and a 3D image constructed.

The use of optical fibers for light delivery in optical microscopes hasbeen established for many years. Throughout this document, the term“fiber” means “optical fiber”. For traditional point scanning confocalmicroscopy, the only fiber that can be used effectively for illuminationof the sample is a single mode fiber. A single mode fiber is a fiberthat is designed for the transmission of a single spatial mode of lightas a carrier. This mode of light may contain a variety of differentwavelengths, although the range of wavelengths that can be transmittedis a function of the cross-sectional dimensions of the core of thefiber. Typical single mode fibers with cores of circular cross sectionhave core diameters only slightly larger than the wavelengths of lightthat they transmit. For example, a fiber that transmits in a band around488 nm has a core diameter of approximately 3.5 μm. Because of the smalldiameter of the fiber core, single mode fibers are used most often withlaser sources. Other sources of radiation are difficult or impossible tocouple into single mode fibers with good efficiency.

The cone angle of light that can be coupled into and is emitted from asingle mode fiber is related to the numerical aperture (NA) of thefiber. The NA of a single mode fiber is a function of the refractiveindices of the fiber core and cladding. The distribution of lightemitted from a single mode fiber is well approximated by a Gaussianshape, the width of which is determined by the NA and by the corediameter of the fiber as well as by the wavelength of the light.

The light that is emitted from the distal end of a single mode fiber maybe considered equivalent to light that is emitted from adiffraction-limited source. This fiber tip is re-imaged through thepinhole and onto the sample at or near its diffraction-limited size.

FIG. 1 illustrates example optics for projecting light from a distal end100 of a single mode fiber 102 with a core diameter D_(F) and numericalaperture NA_(F) (related to the illustrated half-angle θ₁) to a singlepinhole 108 of a diameter D_(P). The optics include a lens 104 having afocal length F₁ and a lens 106 having a focal length F₂. The light exitsthe single mode fiber 102 with a spread of angles given by the numericalaperture NA_(F) of the single mode fiber 102. In general, a numericalaperture NA of a fiber is expressed by Eqn. 1 as:NA=n sin θ,  (1)

where n is a refractive index of the surrounding medium to which thelight exits from the distal end of the fiber, exit angle θ is the angleof divergence of light with respect to an optical axis of the fiber, andsin refers to the trigonometric sine function. In the case that thesurrounding medium is air, the refractive index n is equal to one, thatis n=1. For small angles, θ, and in air, the numerical aperture NA andexit angle θ are approximately equal, that is NA≅θ. The term numericalaperture NA has two definitions when used with fibers. The numericalaperture NA may be defined as a function of the refractive indices ofthe core and cladding or may be defined as in Eqn. 1. In the ray opticsapproximation the two definitions are equivalent. In practice, thenumerical aperture NA as defined by Eqn. 1 is often less than thenumerical aperture NA as defined by the refractive indices of the coreand cladding. Throughout this document, the numerical aperture NA isdefined by Eqn. 1 unless explicitly noted otherwise. By placing thedistal end 100 of the single mode fiber 102 the distance F₁ from thelens 104, light passing through the lens 104 is collimated. A diameterof the lens 104 should be large enough to capture the light emitted fromthe distal end 100 of the fiber 102. By placing the lens 106 at thedistance F₂ from the pinhole 108, the collimated light incident on thelens 106 is focused by the lens 106 through the pinhole 108, asillustrated by half-angle θ₂.

Typically, it is desired in a confocal microscope imaging system for thepinhole spot to be imaged at or near the diffraction limit of themicroscope. To produce the minimum imaged spot size, the lighttransmitted through the pinhole 108 should be the same or larger thanthe largest numerical aperture NA_(MS) of the microscope as measured atthe image plane where the pinhole 108 is located. If the numericalaperture of light exiting the pinhole is larger than numerical apertureNA_(MS) of the microscope, then the minimum imaged spot size can beachieved, however some of the light will be rejected by the microscopeoptics. Ideally, the numerical aperture of light exiting the pinholeshould closely match the numerical aperture NA_(MS) of the microscope sothat the optimum resolution and light transmission to the sample can beachieved. The diameter D_(P) of the pinhole 108 should be chosen so thatthe light exits the pinhole 108 at an angle θ₃ given by the numericalaperture NA_(MS) of the microscope as determined by optical diffractiontheory. That is, NA_(MS) =n sin θ₃.

The focal length F₁ of the lens 104 and the focal length F₂ of the lens106 should be chosen to provide an appropriate magnification M of thelight exiting the single mode fiber 102 so that the focused light on thepinhole 108 just fills the pinhole diameter D_(P). For a diameter D_(F)of light exiting the fiber 102, this magnification M is expressed byEqn. 2 as:

$\begin{matrix}{M = {\frac{D_{P}}{D_{F}} = {- {\frac{F_{2}}{F_{1}}.}}}} & (2)\end{matrix}$

The negative sign indicates that the image of the distal end 100 of thesingle mode fiber 102 at the plane of the pinhole 108 is inverted. Theexact value of the magnification M may be adjusted in practice tofine-tune the trade-off between resolution and light transmission.

In order to achieve both near-diffraction-limited imaging and high lighttransmission to a sample using a point scanning confocal microscope, thefiber used to deliver radiation from the radiation source to themicroscope should be a single mode fiber. If the single mode fiber 102of FIG. 1 was replaced with a larger diameter fiber, the spot sizeproduced at the plane of the pinhole 108 would be too large toefficiently pass through the pinhole 108. While the magnification Mcould be reduced to permit the light to efficiently pass through thepinhole 108, for example, by changing one or both of the focal lengthsF₁ and F₂, this would cause a corresponding increase in the numericalaperture of the light exiting the pinhole 108. This mismatch in thenumerical aperture of the light exiting the pinhole 108 and thenumerical aperture NA_(MS) of the microscope would reduce the amount oflight reaching the sample to be imaged. Alternatively, the pinholediameter D_(P) could be increased to allow more light through thepinhole 108 and more efficient light transmission to the sample withoutchanging the magnification M, but this would result in a larger spotsize and lower resolution. Thus, the use of a non-single mode fiber in asingle point scanning confocal microscope would require either areduction in the system resolution, a reduction in light transmission tothe sample, or a combination of both.

A recent development in optical microscopy has been the parallelapplication of the confocal technique. By the use of various opticalmeans, a plurality of near-diffraction-limited illumination points areprojected onto or into the sample. Each of these points is imagedthrough a corresponding pinhole at a conjugate focal plane onto an imagesensor of a detector, such as a high-sensitivity imaging camera. Ineffect, such a system operates as a plurality of point scanning confocalsystems operating in parallel. Several commercial implementations ofthis concept exist on the market today and can be referred to in generalas multiplexed confocal systems.

One implementation of a multiplexed confocal system uses a spinning diskcomprising a pattern of several thousand pinholes. An example of onesuch spinning disk confocal system is one which comprises a Nipkow disk.The use of a multiplexed confocal system employing the Nipkow diskmethod with microlenses has been disclosed in, for example, U.S. Pat.No. 7,592,582 to Mikuriya et al. The microlenses create a plurality offocal points. A confocal system which creates multiple focal pointsusing microlenses, micromirrors or other focusing elements may bereferred to as a multi-focal confocal system and forms a subset ofmultiplexed confocal systems.

In the instrument described in U.S. Pat. No. 7,592,582, the excitinglaser light is coupled to the incident end of an optical fiber by acondenser lens and is guided by the optical fiber to an inlet of aconfocal scanner unit. A diverging beam of exciting light emitted fromthe distal end of the optical fiber is converted into a collimated beamby a collimating lens. The collimated beam falls on a disk with amicrolens array that focuses excitation laser light onto a pinhole disk(Nipkow disk) mounted on the same axis in such a way that each lensfocuses its light onto a corresponding pinhole. Multiple exciting lightbeams are converged to a sample by an objective lens. Fluorescenceand/or scattered light and/or reflected light originating from thesample passes through the objective lens again, returns through the samepinholes and is reflected by a dichroic mirror positioned between themicrolens disk and the Nipkow disk. The image is then focused onto animage sensor by a relay lens.

In such an apparatus, the Nipkow disk is co-rotated with the microlensdisk at a constant speed, and the converged points of light on thesample are scanned with the pinholes moved by the rotation. A plane ofthe Nipkow disk, a plane to be observed in the sample, and an imagesensor plane are arranged to be conjugate with each other optically.Therefore, an optically sectioned image, that is a confocal image of thesample, is formed on the image sensor. Such a system as described aboveis made by Yokogawa Electric Corporation of Japan and given designationssuch as CSU-10, CSU-21, CSU-22 and CSU-Xl.

Other implementations of multi-focal confocal systems using microlensesexist where the key differences are in the geometry of the microlenspatterns and the scanning mechanisms for moving the microlenses andpinholes. An example of such a system is called the Infinity and isbuilt by VisiTech International Ltd. of Sunderland, United Kingdom.

Illumination methods for multi-focal confocal systems are similar totraditional point scanning systems and, until very recently, have usedsingle mode fibers. In this case, the microlenses image the distal endof the fiber to many parallel pinholes at or near the diffraction limit.As with confocal point scanning systems, the typical radiation sourcefor multi-focal confocal systems is a laser or multiple lasers.

There are disadvantages to using single mode fibers for someapplications. Systems using single mode fibers are, in practice,restricted to radiation sources that emit light with small etendue, suchas lasers with good beam quality, for example, beam quality factorM²<1.2. Laser sources with good beam quality can be coupled to singlemode fibers with coupling efficiencies of approximately 45% to 85%,although the efficiency in practice is sometimes less. Lasers withlesser beam quality couple with even lower efficiencies. Single modefibers can only operate as such over a limited spectral range. Above agiven upper cutoff wavelength the fiber core is too small to transmitlight with low losses. Below a lower cutoff wavelength, the light is nolonger transmitted in a single mode. The Gaussian distribution of thesingle mode fiber output intensity is less than ideal for systemsrequiring even illumination. Only the central part of the Gaussian beamis often used, such that the variation in intensity is less than someamount, for example 20%. In such systems a compromise between uniformityin light distribution across an image plane and the light utilizationefficiency is required because the peripheral part of the Gaussian beamis abandoned.

Another disadvantage of systems that use single mode fibers is therequirement for high thermal, mechanical, and temporal stability of thelaser-to-fiber alignment and the high manufacturing cost of such stablesystems. Designing a means of providing stable laser-to-fiber coupling,and the creation of systems coupling multiple lasers to a single modefiber, can be challenging.

As an alternative to using single mode fibers for delivery of radiationin optical microscopes, the use of multimode fibers has recently beencontemplated. A multimode fiber is an optical fiber that is designed tocarry multiple light ray paths or modes concurrently over a broadspectrum of wavelengths. It can be thought of simply as a long lighttube. The use of a multimode fiber may reduce the sensitivity of thecoupling between the radiation source and fiber to mechanical andtemperature influences, thereby enabling a variety of radiation sourcesand wavelengths to be used for illumination in an optical microscope.

In “A Mercury Arc Lamp-Based Multi-Color Confocal Real Time ImagingSystem for Cellular Structure and Function”, Cell Structure andFunction, vol. 3, pages 133-141, 2008), Saito et al. describes the useof a multimode fiber with a 1 mm core diameter to couple an arc lamp toa Yokogawa CSU-10. The efficiency of the light coupled from the end ofthe multimode fiber through the CSU is reported to be 1%. While it wasnot clearly defined how this measurement was made, this numberrepresents a low efficiency of light utilization. Saito et al. do notuse this fiber with a laser but only with a broadband arc lamp source.Furthermore, with the use of such a large-diameter fiber, much of thelost light is scattered from the back surface of the pinhole disk, thusleading to a higher potential for a loss of contrast.

Use of a multimode fiber to efficiently deliver light emitted from aradiation source to a multi-focal confocal microscope has been disclosedby Berman in U.S. Patent Publication 2010/0142041. Berman discloses amethod of selecting a core diameter and a numerical aperture of amultimode fiber such that light emitted from a distal end of themultimode fiber is transmitted through the confocal pinhole array withreasonable efficiency.

Typically, the intensity of light emitted from a distal end of amultimode fiber decreases at points further from the optical axis of amultimode fiber in the transverse plane. Therefore, a trade-off is madebetween light utilization efficiency and uniformity of illumination ofthe microscope sample. This trade-off may be realized by limiting thesample illumination to light from the central area of the collimatedbeam. Illuminating a smaller area may result in more uniformillumination but may use a smaller fraction of the light from themultimode fiber. Illuminating a larger area may result in better lightutilization efficiency but may reduce the uniformity of theillumination.

SUMMARY

In the distant past, when light sources were weak, a filament was usedto illuminate a sample to be imaged by a microscope. If the filament wasimaged onto the sample plane, the intensity profile of the imagemirrored the intensity profile of the filament. Known as “criticalillumination”, this was useful for achieving very bright illumination ona very small spot, but had limited applications due to its inability toprovide uniform illumination of the entire sample or of a portion of thesample that is larger than a very small spot. More traditionally, theoptical system coupling the filament to the sample plane was used todefocus the illumination, yielding a weaker but more uniformillumination of the sample. This is known as “Köhler illumination.” Inview of the desire for substantially uniform illumination of a sample tobe imaged, microscope systems using optical fibers for delivery of lightcontinue to be configured for Köhler illumination, so that the distalend of the optical fiber is defocused at the sample plane.

As described in more detail below, the inventor proposes imaging thedistal end of a multimode fiber onto a sample plane. A sample plane is aplane in a sample to be probed by a microscope. Where a transversedistribution of light exiting the distal end of the multimode fiber issubstantially uniform, a radiant intensity of the light forming theimage of the distal end at the sample plane is also substantiallyuniform. The radiant intensity of the light at the sample plane issubstantially uniform as long as the distal end is imaged sufficientlyclose to the sample plane, even if the distal end is not imagedprecisely onto the sample plane. Substantially uniform illumination of asample to be probed by a microscope is therefore achievable where (i)the transverse distribution of light exiting the distal end of themultimode fiber is substantially uniform and (ii) a light-coupling unitthat couples the multimode fiber to the microscope is configured toimage the distal end of the multimode fiber onto or sufficiently closeto the sample plane. Persons of ordinary skill in the art willappreciate that substantially uniform illumination of a sample is alsoachievable when, instead of or in addition to imaging the distal end ofthe multimode fiber onto or sufficiently close to the sample plane, thelight-coupling unit images the distal end of the multimode fiber onto orsufficiently close to a plane that is optically conjugate to the sampleplane.

The efficiency of the illumination may be improved, for example, byusing a light-coupling unit that is further configured to provide amagnification such that an area of substantially uniform illumination onthe sample plane is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of a detector, such asa high-sensitivity imaging camera. The active region of the detector maybe understood to be that portion of an image sensor within the detectorthat may be activated by light received from anywhere within a field ofview of the microscope.

As described in more detail below, for the case of a multi-focalconfocal subsystem that comprises a segmented focusing array and anillumination aperture array, the inventor proposes imaging the distalend of a multimode fiber onto the plane of the segmented focusing array.The illumination aperture array is imaged to the sample plane. Where atransverse distribution of light exiting the distal end of the multimodefiber is substantially uniform, a radiant intensity of the light formingthe image of the distal end at the plane of the segmented focusing arrayis also substantially uniform. The radiant intensity of the light at theplane of the segmented focusing array is substantially uniform as longas the distal end is imaged sufficiently close to the plane of thesegmented focusing array, even if the distal end is not imaged preciselyonto the plane of the segmented focusing array. Substantially uniformillumination of the sample after a complete scan of the illuminationapertures is therefore achievable where (i) the transverse distributionof light exiting the distal end of the multimode fiber is substantiallyuniform and (ii) a light-coupling unit in the multi-focal confocalsubsystem that couples the multimode fiber to the segmented focusingarray is configured to image the distal end of the multimode fiber ontoor sufficiently close to the plane of the segmented focusing array.

The efficiency of the illumination may be improved, for example, byusing a light-coupling unit that is further configured to provide amagnification such that an area of substantially uniform illumination onthe plane of the segmented focusing array is not substantially biggerthan an imaged area of the sample plane that is imaged by any activeregion of a detector, such as a high-sensitivity imaging camera. Theactive region of the detector may be understood to be that portion of animage sensor within the detector that may be activated by light receivedfrom anywhere within a field of view of the microscope. The efficiencyof the illumination may be improved, for example, where the multimodefiber, the light-coupling unit, the segmented focusing array and theillumination aperture array are configured such that light exiting thedistal end of the multimode fiber parallel to an optical axis of themultimode fiber is focused by focusing elements of the segmentedfocusing array onto the centers of the corresponding illuminationapertures. The efficiency of the illumination may be improved, forexample, where the dimensions of the cross section of the core of themultimode fiber and the numerical aperture of the multimode fibersatisfy a particular relationship to the numerical aperture NA_(MS) ofthe microscope and properties of the segmented focusing array and theillumination aperture array.

The multimode fiber may have a core of circular cross section, squarecross section, rectangular cross section, or any other suitable crosssection. A step-index multimode fiber is an example of a multimode fiberfor which the transverse distribution of light exiting the distal end issubstantially uniform. It is contemplated that other multimode fibersalso exhibit the behavior that the transverse distribution of lightexiting the distal end is substantially uniform. In the expression“transverse distribution of light exiting the distal end of themultimode fiber”, the term “transverse” means transverse to an opticalaxis of the multimode fiber.

The methods and systems described herein may have a wide variety ofapplications in other areas of optical microscopy, including, but notlimited to, wide field and bright field illumination, fluorescencerecovery after photobleaching (FRAP), fluorescence lifetime imaging(FLIM), structured illumination (SIM), photo-activated localizationmicroscopy (PALM) and stochastic optical reconstruction microscopy(STORM).

BRIEF DESCRIPTION OF THE DRAWINGS

The technology described herein is illustrated by way of example and notlimitation in the figures of the accompanying drawings, in which likereference numerals indicate corresponding, analogous or similarelements, and in which:

FIG. 1 is a schematic showing an example set of optics to couple anoptical fiber to a single pinhole as in a point scanning confocalmicroscope, as known in the art;

FIG. 2 is a simplified block diagram showing an example microscopesystem;

FIG. 3 is a simplified block diagram showing an example multi-focalconfocal microscope system;

FIG. 4-1 is a simplified cross-sectional diagram of an examplestep-index optical fiber with a circular core;

FIG. 4-2 is a simplified cross-sectional diagram of an examplestep-index optical fiber with a square core;

FIG. 4-3 is a simplified cross-sectional diagram of an examplestep-index optical fiber with a rectangular core;

FIG. 5 is a schematic showing a first set of example paths of light raysin a multi-focal confocal subsystem coupled to a multimode fiber;

FIG. 6-1 is a schematic showing a second set of example paths of lightrays in a multi-focal confocal subsystem coupled to a multimode fiber;and

FIG. 6-2 is a schematic showing a third set of example paths of lightrays in a multi-focal confocal subsystem coupled to a multimode fiber.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity.

DETAILED DESCRIPTION

An “illumination aperture”, as used herein, refers to any illuminationaperture that is suitable for use in confocal microscopy, as would beapparent to someone skilled in the art. For example, an “illuminationaperture array” refers to a plurality of pinholes, a plurality of slits,a Nipkow array, or any other suitable plurality of illuminationapertures. Any suitable plurality of illumination apertures can besubstituted in the examples described below for the pinhole array ofpinholes employed in the examples.

A “segmented focusing array of focusing elements”, as used herein,refers to any plurality of optical elements that can be used to focuslight onto corresponding illumination apertures of an illuminationaperture array. The optical elements may be microlenses, micromirrors,or any other focusing elements, including reflective focusing elementsand diffractive focusing elements, as would be apparent to someoneskilled in the art. Any segmented focusing array of focusing elementscan be substituted in the examples described below for the microlensarray of microlenses employed in the examples.

The terms “light” and “radiation” may be used interchangeably and referto radiation in the UV-visible-NIR (ultraviolet-visible-near infrared)spectral range. The term “radiation source” may refer to any source ableto generate and emit radiation, including but not limited to, lightemitting diodes (LEDs), solid state devices, super luminescent diodes(SLDs), arc lamps, or any other suitable radiation sources as would beapparent to someone skilled in the art.

As used herein, a “microscope” comprises at least an objective. A“microscope system” is a system that may be used to probe a sample byprojecting light or radiation onto the sample, thus producing reflectedlight or scattered light or fluorescence light or any combinationthereof from the sample. As used herein, “multiplexed confocalmicroscopy” refers to the use of a plurality of illumination aperturesto apply the confocal technique in parallel to a microscope. As usedherein, “multi-focal confocal microscopy” refers to the use of asegmented focusing array of focusing elements in multiplexed confocalmicroscopy.

FIG. 2 illustrates an example of an optical microscope system 200comprising a radiation source module 202, a radiation delivery module204 and a microscope module 206.

The radiation source module 202 generates and optionally conditionsradiation for acceptance into a multimode fiber 208 of the radiationdelivery module 204. The example radiation source module 202 comprises aradiation source 210 emitting light of one or more wavelengths, followedby a light control and conditioning unit 212, a light combining unit 214and a light-coupling unit 216.

The radiation source 210 may comprise one or more individual radiationsources 218. The radiation sources 218 are provided with one or morepower supplies (not shown) and may generate radiation of one or morewavelengths. An example implementation of an optical microscope systemuses lasers as the radiation sources 218 but other implementations coulduse any radiation sources emitting light that can be coupled to themultimode fiber 208.

Optics of the light control and conditioning unit 212 are designed toprovide the radiation beam being guided into an incident end 220 of themultimode optical fiber 208 by a lens 222 with a circular cross sectionof a predetermined diameter. The light control and conditioning unit 212may comprise one or more light control and conditioning subunits 224,each of the light control and conditioning subunits 224 corresponding toone of the individual radiation sources 218. For the sake of simplicity,each of the light control and conditioning subunits 224 is shownschematically as a simple Galilean telescopic beam expander that isbuilt from a negative lens 226 and a positive lens 228. Alternativeimplementations of the light control and conditioning subunits 224 maycomprise anamorphic prismatic or cylindrical optics to provide anelliptical laser beam with the circular property and/or beam-shapingmeans to make adjustments enabling the light intensity distribution tobe more uniform (such as the means disclosed in U.S. Pat. No. 7,592,582for converting a beam of light outgoing from a distal end of an opticalfiber into a collimated beam with a predetermined intensitydistribution, for example). The light control and conditioning subunits224 may comprise additional beam shaping means to enable the lightintensity distribution to be more uniform or to get a predeterminedintensity profile at a distal end 230 of the multimode fiber 208. Suchadditional beam shaping means will be known to someone skilled in theart.

The light control and conditioning subunits 224 may optionally comprisemeans for control of individual radiation source power and/or wavelengthto optimize micro-imaging conditions (not shown). For example, in thecase that one of the individual radiation sources 218 is a laser, thecorresponding one of the light control and conditioning subunits 224 maycomprise an acousto-optic tunable filter (AOTF), or alternatively amechanical shutter followed by a continuously variable neutral densityfilter wheel. The use of additional light control means will be known tosomeone skilled in the art.

The light combining unit 214 comprises optical elements that combinecollimated beams of different wavelengths from the light control andconditioning unit 212 and direct them to the light-coupling unit 216.The light combining unit 214 may comprise optics of any form, and mayinclude, for example, one or more folding mirrors 232, dichroic mirrors234, and any other suitable optical elements, as would be apparent tosomeone skilled in the art.

The light-coupling unit 216 may comprise a lens 222 designed to focusthe multi-wavelength collimated beam of a predetermined diameter ontothe incident end 220 of the multimode optical fiber 208. The lens 222may have a short focal length, and may be a collimator lens, a condenserlens, a micro-objective, or some other suitable lens as would beapparent to someone skilled in the art. The cross-sectional diameter ofthe multi-wavelength collimated beam and the focal length of the lens222 may be selected or designed to obtain a specific numerical apertureof the input focused beam that is proportional and close to a resultingnumerical aperture of a beam emitted from the distal end 230 of themultimode fiber 208.

The incident end 220 and the distal end 230 of the multimode fiber 208may be connected or connectable to an input connector and an outputconnector, respectively. The input and output connectors (not shown) maybe of any type, for example, fixed connection (FC) type connectors, orany other suitable connectors as would be apparent to someone skilled inthe art.

A transverse distribution of light exiting the multimode fiber 208 issubstantially uniform. The core of the multimode fiber 208 may have acircular cross section, a square cross section, a rectangular crosssection, or any other suitable cross section. The use of a multimodefiber having a core of square cross section or a core of rectangularcross section may have an advantage in that the illumination area of asample to be imaged can be better matched to the shape of an imagesensor of a detector. The use of a multimode fiber having a core ofcircular cross section may have an advantage of being less expensive dueto its greater availability. Examples of multimode fibers will bedescribed in greater detail with respect to FIG. 4.

For example, the multimode fiber 208 is a step-index fiber. A step-indexfiber has a constant refractive index in its core and a step to a lowerrefractive index at the core-cladding interface. Many types ofstep-index fibers may produce a substantially uniform transverseintensity distribution of light exiting the distal end 230 of themultimode fiber 208. The quality of the light uniformity is related tothe material used in the core and cladding and to the manufacturingprocess.

The radiation delivery module 204 may comprise additional elements notshown in FIG. 2. For example, the radiation delivery module may comprisemeans for phase randomization or additional optical fibers or both. Someexamples of these elements will be described in greater detail withrespect to FIG. 3.

The microscope module 206 comprises a light-coupling unit 236,light-splitting optics 254, a microscope 238, and a light-detecting unit240.

The light-coupling unit 236 is shown in FIG. 2 to comprise a lens 242and a lens 244, but may also comprise various other optical elements(not shown). A non-exhaustive list of examples of alternative oradditional elements comprised in the light-coupling unit 236 includes asingle lens, multiple lenses, mirrors, multiple mirrors or anycombination of suitable optical elements.

The microscope 238 comprises an objective 246 focusing light receivedfrom the light-coupling unit 236 onto a sample 248 at a sample plane250. Any suitable objective 246 may be used as would be apparent tosomeone skilled in the art. The microscope 238 may also compriseadditional elements (not shown), as would be apparent to someone skilledin the art.

The light splitting optics 254 are designed to pass exciting radiationreceived from the light-coupling unit 236 to the sample 248 via theobjective 246 of the microscope 238, and to reflect return light fromthe sample 248 to the light-detecting unit 240. The return light isoften produced by fluorescence but can result from reflection, Ramanscattering or any other elastic or inelastic light scattering effect aswould be known to someone skilled in the art.

A non-exhaustive list of examples of the light splitting optics 254includes a dichroic mirror, a splitter based on polarization, a mirrorwith a partially reflective surface, or any other device to divide theexcitation light from the return light as would be apparent to someoneskilled in the art.

The light-detecting unit 240 comprises light-detecting optics 256 and adetector 258, such as a high-sensitivity imaging camera. Thelight-detecting optics 256 may comprise a relay lens 260, and maycomprise additional elements (not shown), such as a blocking filterand/or narrow band filters for multi-spectral imaging. A non-exhaustivelist of examples of the detector 258 includes a charge-coupled device(CCD) camera, a complementary metal-oxide semiconductor (CMOS) camera,an intensified CCD (ICCD) camera, and any other suitable camera as wouldbe apparent to someone skilled in the art. A 3CCD camera with additionalnarrowband filters may be applied for simultaneous multi-spectralimaging.

The sample 248 and the detector 258 may be arranged such that the sampleplane 250 and a plane of an image sensor of the detector 258, also knownas the image sensor plane (not shown), are optically conjugate with eachother in order to provide an image of the sample 248. The portion of theimage sensor plane that may be activated by light received from anywherewithin a field of view of the microscope is known as the active regionof the detector 258.

The light-coupling unit 236 is configured to image the distal end 230 ofthe multimode fiber 208 onto the sample plane 250 or onto a plane 252that is optically conjugate to the sample plane 250. Alternatively, thelight-coupling unit 236 is configured to image the distal end 230 of themultimode fiber 208 sufficiently close to the sample plane 250 orsufficiently close to the conjugate plane 252 such that a radiantintensity of light at the sample plane 250 or the conjugate plane 252 issubstantially uniform. Such configuration of the light-coupling unit 236may result in substantially uniform illumination of the sample 248.

The light-coupling unit 236 may be configured to provide a magnificationsuch that the area of substantially uniform illumination on the sampleplane 250 is not substantially bigger than an imaged area of the sampleplane that is imaged by any active region of the detector 258. This mayimprove the efficiency of the illumination.

FIG. 3 illustrates an example of a multi-focal confocal microscopesystem 300 comprising the radiation source module 202, a radiationdelivery module 304 and a microscope module 306.

As discussed with respect to FIG. 2, the radiation delivery module 204may comprise additional elements beyond the multimode fiber 208. In thecase that the radiation source 210 generates coherent radiation, forexample, if one or more of the individual radiation sources 218 is alaser, the radiation delivery module 204 may further comprise a phaserandomizer. A phase randomizer is designed to suppress speckle that ispresent due to temporal coherence properties of coherent radiation.

The radiation delivery module 304 shown in FIG. 3 comprises a phaserandomizer in the form of a vibrating mechanical driver 308 designed togenerate vibrations in a section of the multimode fiber 208. Thevibrating mechanical driver 308 may be mechanically coupled to thesection of the multimode fiber 208, for example, by being attached to aportion of the multimode fiber 208 that is coiled and loosely held byretaining clamps, allowing a long length of the multimode fiber 208 tobe vibrated. The vibrations induced by the vibrating mechanical driver308 may result in fast variations of the optical path lengths ofindividual rays in the multimode fiber 208, randomizing the coherenceeffects of the light, and as a result, suppressing spatial modulation ofthe amplitude of image of the sample (speckle). The vibrating mechanicaldriver 308 may be built in accordance with Ellis et al. (J. Cell Biol.83:303a, 1979) and/or may include a piezoelectric driver, or anyelectromechanical setup able to generate suitable vibrations as would beapparent to someone skilled in the art. For example, the vibratingmechanical driver 308 may be built in the form of an electromotor withan unbalanced weight fixed on its shaft or a linear voice coil motor.

Although not explicitly shown, a phase randomizer may alternatively oradditionally comprise a rotating diffuser placed in proximity to theincident end 220 of the multimode fiber 208 or to the distal end 230 ofthe multimode fiber 208. The rotating diffuser could also be placed at aconjugate image plane of the distal end 230 of the multimode fiber 208or at a conjugate image plane of the incident end 220 of the multimodefiber 208. The rotating diffuser may comprise a high-speed electromotorto rotate the diffuser.

To avoid high optical power losses when using a rotating diffuser, thedistance z_(d) traveled by the light from the rotating diffuser to theincident end 220 of the multimode fiber 208, or the distance z_(d)traveled by the light from the distal end 230 of the multimode fiber 208to the rotating diffuser should satisfy Eqn. 3:

$\begin{matrix}{{z_{d} < \frac{D_{F}}{\theta_{d}}},} & (3)\end{matrix}$where D_(F) is the core diameter of the fiber (in the case of a fiberhaving a circular cross section) and θ_(d) is a FWHM (full width at halfmaximum) of the angular light distribution of the rotating diffuser.

Although not explicitly shown, it is further contemplated that theradiation delivery module 304 may comprise one or more optical fibers inaddition to the multimode fiber 208. The additional optical fibers maybe multimode fibers or single mode fibers and may have input and outputconnectors of any type, for example, FC type connectors, or any othersuitable connectors as would be apparent to someone skilled in the art.The additional optical fibers may be used in conjunction with themultimode fiber 208 to deliver radiation from the radiation sourcemodule 202 to the microscope module 306. For example, an additionaloptical fiber may receive radiation from the radiation source module202, which it then transmits to the multimode fiber 208 via a rotatingdiffuser of a phase randomizer. In this case, the phase randomizer isdesigned to randomize the phase of coherent radiation as the radiationis transmitted from the optical fiber to the multimode fiber 208. Inanother example, a vibrating mechanical driver 308 may be used torandomize the phase of light delivered to the microscope module 306 byinducing vibrations in a section of the multimode fiber 208 or in asection of an additional optical fiber.

Other types of speckle reduction may be employed as alternatives or inaddition to the fiber disturbance through a vibrating mechanical driveror a rotating diffuser. One such example is the movement or vibration ofa tip of the multimode fiber 208.

To achieve the multi-focal confocal effect, the microscope module 306has a microlens array disk 310 comprising a plurality of individualmicrolenses 312, and a pinhole array disk 314 comprising a plurality ofpinholes 316. The light-coupling unit 236, the microlens array disk 310,the light splitting optics 254, and the pinhole array disk 314 togetherform a multi-focal confocal subsystem 318.

The pinhole array disk 314 is mounted on a same axis of the microlensarray disk 310 at a distance from the microlens array disk 310 that issubstantially equal to a focal length of the microlenses 312, in such away that each microlens 312 focuses its light onto a differentindividual pinhole 316 comprised in the pinhole array disk 314. Thepinhole array disk 314 is co-rotated with the microlens array disk 310at a constant speed.

In order to provide a confocal image of the sample 248, the pinholearray disk 314, the sample 248, and the detector 258 may be arrangedsuch that a plane 352 of the pinhole array disk 314, the sample plane250, and the image sensor plane (not shown) are optically conjugate witheach other.

As discussed with respect to FIG. 2, a transverse distribution of lightexiting the multimode fiber 208 is substantially uniform. The core ofthe multimode fiber 208 may have a circular cross section, a squarecross section, a rectangular cross section, or any other suitable crosssection. The use of a multimode fiber having a core of square crosssection or a core of rectangular cross section may have an advantage inthat the illumination area of a sample to be imaged can be bettermatched to the shape of an image sensor of a detector. The use of amultimode fiber having a core of circular cross section may have anadvantage of being less expensive due to its greater availability.Examples of multimode fibers will be described in greater detail withrespect to FIG. 4.

For example, the multimode fiber 208 is a step-index fiber. A step-indexfiber has a constant refractive index in its core and a step to a lowerrefractive index at the core-cladding interface. Many types ofstep-index fibers may produce a substantially uniform transverseintensity distribution of light exiting the distal end 230 of themultimode fiber 208. The quality of the light uniformity is related tothe material used in the core and cladding and to the manufacturingprocess.

The light-coupling unit 236 is configured to image the distal end 230 ofthe multimode fiber 208 onto the microlens array plane 352.Alternatively, the light-coupling unit 236 is configured to image thedistal end 230 of the multimode fiber 208 sufficiently close to themicrolens array plane 352 such that a radiant intensity of light at themicrolens array plane 352 is substantially uniform. Such configurationof the light-coupling unit 236 may result in substantially uniformillumination of the sample 248 after a complete scan of the pinholes.

The light-coupling unit 236 may be configured to provide a magnificationthat is large enough such that the size of the image of the distal end230 of the multimode fiber 208 at the microlens array plane 352 issufficient to illuminate with substantial uniformity all the pinholes316 that are used by the detector 258 to construct the confocal image ofthe sample 248. The light-coupling unit 236 may be further configured toprovide a magnification such that the area of substantially uniformillumination at or near the sample plane 250 after a complete scan ofthe pinholes is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of the detector 258.This further configuration of the light-coupling unit 236 may improvethe efficiency of the illumination. An example of a suitableconfiguration of optical elements for substantially uniform andefficient illumination is described with respect to FIGS. 5 and 6.

FIGS. 4-1, 4-2, and 4-3 illustrate cross-sectional diagrams of examplestep-index optical fibers, together with a corresponding plot ofrefractive index as a function of position along a diameter of thefiber.

FIG. 4-1 illustrates a cross section of an optical fiber 400 having acore 402 of circular cross section of diameter D_(F) and a cladding 404surrounding the core 402. The cladding 404 may have a circular outercross section as shown, or any other suitable cross section. Thecladding 404 has a refractive index n₁ and the core 402 has a refractiveindex n₂, where the refractive index n₁ is less than the refractiveindex n₂, that is n₁<n₂. The plot of refractive index of the opticalfiber 400 is shown below the optical fiber 400 as a function of positionalong a dashed line 406, assuming the optical fiber 400 is surrounded byair (refractive index n=1). It is also contemplated that the cladding404 has a non-uniform refractive index, as long as the refractive indexat the boundary of the core 402 and the cladding 404 changes abruptlyfrom n₂ to n₁.

FIG. 4-2 illustrates a cross section of an optical fiber 408 having acore 410 of square cross section of width D_(F) and height D_(F) and acladding 412 surrounding the core 410. The cladding 412 may have acircular outer cross section as shown, or it may have any other suitablecross section. The cladding 412 has a refractive index n₁ and the core410 has a refractive index n₂, where the refractive index n₁ is lessthan the refractive index n₂, that is n₁<n₂. The plot of refractiveindex of the optical fiber 408 is shown below the optical fiber 408 as afunction of position along a dashed line 414, assuming the optical fiber408 is surrounded by air (refractive index n=1). It is also contemplatedthat the cladding 412 has a non-uniform refractive index, as long as therefractive index at the boundary of the core 410 and the cladding 412changes abruptly from n₂ to n₁.

FIG. 4-3 illustrates a cross section of an optical fiber 416 having acore 418 of rectangular cross section with a width D_(FW) and heightD_(FH) and a cladding 420 surrounding the rectangular core 418. Thecladding 420 may also have an elliptical outer cross section as shown,or it may have any other suitable cross section. The cladding 420 has arefractive index n₁ and the rectangular core 418 has a refractive indexn₂, where the refractive index n₁ is less than the refractive index n₂,that is n₁<n₂. The plot of refractive index of the optical fiber 416 isshown below the optical fiber 416 as a function of position along adashed line 422, assuming the optical fiber 416 is surrounded by air(refractive index n=1). It is also contemplated that the cladding 420has a non-uniform refractive index, as long as the refractive index atthe boundary of the core 418 and the cladding 420 changes abruptly fromn₂ to n₁.

The numerical aperture NA_(F) of an optical fiber is related to thedifference between the refractive index n₁ of the cladding and therefractive index n₂ of the core. A particular step-index multimode fibermight have a cladding of refractive index n₁=1.46 and a core ofrefractive index n₂=1.48, with both the core and the cladding made of asilica composition. In terms of dimensions, the core might have acircular cross section of diameter D_(F)=200 μm, and the length of themultimode fiber might be 2 meters. It should be noted that theseproperties are merely examples, and many other step-index multimodefibers are possible.

Example optics, configured to image a distal end of a multimode fiberonto a microlens array of a multi-focal confocal subsystem, will now bedescribed with respect to FIGS. 5, 6-1 and 6-2. Where a transversedistribution of light exiting the distal end of the multimode fiber issubstantially uniform, such configuration of the optics may produce asubstantially uniform radiant intensity on the microlenses of themicrolens array, thus making it possible to achieve a substantiallyuniform intensity of light transmitted through corresponding pinholes ofa pinhole array, which, after a complete scan of the pinholes, mayresult in substantially uniform illumination of a sample to be probed bya microscope. Furthermore, as explained in detail below, by selecting anappropriate step-index multimode fiber and light-coupling unit, it maybe possible to achieve efficient transmission of light emitted from thedistal end of the multimode fiber through the pinholes.

FIG. 5 illustrates example optics projecting light from a distal end 500of a multimode fiber 502 through lenses 504 and 506 and onto a microlensarray 508. The microlens array 508 comprises a plurality of individualmicrolenses 510 which focus the light from the distal end 500 onto apinhole array 512 comprising a plurality of individual pinholes 514. Themultimode fiber 502 is an example of the multimode fiber 208 illustratedin FIGS. 2 and 3, and therefore a transverse distribution of lightexiting the distal end of the multimode fiber 502 is substantiallyuniform. The microlens array 508 and the pinhole array 512 are examplesof the microlens array disk 310 and the pinhole array disk 314,respectively, illustrated in FIG. 3. In this example, the multimodefiber 502 is a step-index multimode fiber with a numerical apertureNA_(F) and has a core of either circular cross section, square crosssection, or rectangular cross section.

Lenses 504 and 506 are example optical elements that may be comprised inthe light-coupling unit 236 described with respect to FIG. 3. The lenses504 and 506 are positive focal length achromatic doublets with focallengths F₃ and F₄, respectively. This type of lens may be desirable forits inexpensiveness and ability to handle aberrations. However, the useof alternative and/or additional optical elements in the light-couplingunit 236 is contemplated.

The distal end 500 of the multimode fiber 502 is placed a distance U₁from the lens 504, and the microlens array 508 is placed a distance U₂from the lens 506. The lenses 504 and 506 are separated by a distance U₃equal to a sum of their focal lengths F₃ and F₄, that is U₃=F₃+F₄,thereby forming an optical relay. The microlens array 508 and thepinhole array 512 are separated by distance U₄ substantially equal to afocal length F₅ of the microlenses 510, that is U₄=F₅, so that lightfocused by the microlenses 510 is focused on the pinhole array 512.

Using the thin lens approximation, the lens system of the lens 504 andthe lens 506 images the distal end 500 of the multimode fiber 502 with amagnification M of:

$\begin{matrix}{M = {- {\frac{F_{3}}{F_{4}}.}}} & (4)\end{matrix}$

In order for the lens system of the lens 504 and the lens 506 to imagethe distal end 500 of the multimode fiber 502 onto the microlens array508, and using the thin lens approximation and paraxial approximations,the distance U₂ between the lens 506 and the microlens array 508, isexpressed by Eqn. 5 as:

$\begin{matrix}{U_{2} = {\left\lbrack {F_{3}^{2} + {F_{3}F_{4}} - {U_{1}F_{4}}} \right\rbrack{\frac{F_{4}}{F_{3}^{2}}.}}} & (5)\end{matrix}$

where Eqn. 5 takes into account the distance U₃=F₃+F₄ between the lens504 and the lens 506.

In the typical case where the distal end 500 of the multimode fiber 502is positioned such that the distance U₁ between the distal end 500 ofthe multimode fiber 502 and the lens 504 is substantially equal to thefocal length F₃ of the lens 504, that is U₁=F₃, the distance U₂ betweenthe lens 506 and the microlens array 508 determined by Eqn. 5 is equalto the focal length F₄ of the lens 506, that is U₂=F₄. Small adjustmentsmay be made to the distance U₁ to allow greater flexibility inpositioning the image of the distal end 500 of the multimode fiber 502on the microlens array 508. Eqn. 5 is valid in the thin lens andparaxial approximations for optical systems. Adjustments that may beneeded for small deviations from these approximations are obvious tothose skilled in the art.

Using the paraxial ray approximation, a light ray 516 originating fromthe center of the distal end 500 of the multimode fiber 502 at an angleθ₁ relative to an optical axis 518 of the multimode fiber 502 will gothrough the center of a central microlens 520 at an angle θ₂ relative tothe optical axis 518, where the angle θ₂ is expressed as:

$\begin{matrix}{\theta_{2} \cong {\frac{\theta_{1}}{M}.}} & (6)\end{matrix}$

The light ray 516 will then pass through a corresponding one of thepinholes 514 at a distance from the center of the pinhole 514 equal to aproduct of the focal length F₅ of the microlenses 510 and the angle θ₂,that is F₅θ₂. In order for light to pass efficiently from the pointcenter of the distal end 500 of the multimode fiber 502 through thepinhole 514 corresponding to the central microlens 520, this system ofthe multimode fiber 502, the lens 504, the lens 506, the microlens array508 and the pinhole array 512 should meet the requirement expressed byEqn. 7:

$\begin{matrix}{D_{P} \geq {2\; F_{5}\theta_{2}} \cong {\frac{2\; F_{5}\theta_{1}}{M}.}} & (7)\end{matrix}$

The diameter D₅ and the focal length F₅ of the microlenses 510 aretypically designed in a multi-focal confocal subsystem to produce exitlight from the pinhole 514 that is at or near the designed acceptancenumerical aperture NA_(MS) of a microscope when the microlenses 510 areilluminated with collimated light. The approximate relationship betweenthe diameter D₅ and the focal length F₅ of the microlenses 510 is then:2F ₅ NA _(MS) ≅D ₅  (8)

In practice, the relationship of Eqn. 8 may be somewhat different due togeneralization of the multi-focal confocal subsystem to work with avariety of microscopes and objectives. For instance, the numericalaperture of the microlenses 510 may not match the numerical apertureNA_(MS).

FIGS. 6-1 and 6-2 illustrate the paths of light rays in the example ofFIG. 5 that are imaged to one of the microlenses 510 at a distance fromthe optical axis 518. The distance is approximately equal to a productof the number of microlenses between the microlens being considered andthe optical axis, and the diameter of each microlens 510. This distanceis not exact, as the microlenses 510 are typically arranged in a Nipkowarray when viewed in a two dimensional plane, however this approximationis sufficient for the discussion that follows.

FIG. 6-1 illustrates the light rays from the multimode fiber 502 thatare imaged to a center of the Nth microlens 602 in the microlens array508. FIG. 6-2 illustrates light rays that pass through an edge of theNth microlens 602 in the microlens array 508.

Light rays parallel to the optical axis 518, for example rays 604 and606, will remain parallel to the optical axis after passing through thelens 506 as long as the lens 504 and the lens 506 are separated by adistance U₃ equal to a sum of their focal lengths, that is U₃=F₃+F₄.This is true regardless of the distance U₁ between the distal end 500 ofthe multimode fiber 502 and the lens 504. Any ray parallel to theoptical axis 518 will be focused to the center of the pinhole behind themicrolens through which the ray passes, as is shown by rays 604 and 606,if the centers of the pinholes are aligned with the centers of themicrolenses.

A point 608 on the distal end 500 of the multimode fiber 502 is imagedto the center of the Nth microlens 602 from the optical axis 518, wherethe center is a distance D_(i1) the optical axis 518 equal to a productof N and a diameter D₅ of the microlenses 510, that is D_(i1)=N*D₅. Ifthe point 608 is at a distance D_(s1) from the optical axis 518, thenthe relationship between the distance D_(i1) and the distance D_(s1) isexpressed as:D _(i1) ≅|M|D _(s1) ≅ND ₅  (9)

As described above with respect to the light ray 516 and Eqn. 6, a lightray 610 originating from the point 608 at an angle θ₁ relative to theoptical axis 518 will go through the center of the Nth microlens 602 atan angle θ₂≈θ₁|M|. The light ray 610 will then pass through the pinholethat corresponds to the Nth microlens 602, at a distance F₅θ₂ from thecenter of that pinhole. The relation of Eqn. 7 still holds for the lightto efficiently pass through the pinhole that corresponds to the Nthmicrolens 602 from the point 608.

As illustrated in FIG. 6-2, a point 612 on the distal end 500 of themultimode fiber 502 is imaged to the edge of the Nth microlens 602 inthe microlens array 508, and the edge is a distance D_(i2) from theoptical axis 518, where D_(i2) is equal to (N+½)*D₅. If the point 612 isat a distance D_(s2) from the optical axis 518, then the relationshipbetween the distance D_(i2) and the distance D_(s2) is expressed as:D _(i2) ≅|M|D _(s2)≅(N+½)D ₅.  (10)

The angle θ₂ relative to the optical axis 518 of a ray 614 going intothe Nth microlens 602 is still given by Eqn. 6 and the relation of Eqn.7 still holds for the light to efficiently pass through the pinhole thatcorresponds to the Nth microlens 602 from the point 612.

The magnification M provided by the system of lenses 504 and 506 may bechosen so that the dimensions D_(x) and D_(y) of the image of the distalend 500 of the multimode fiber 502 on the microlens array 508 are largeenough to illuminate all the pinholes 514 that are used to construct theconfocal image. If there is a total of N_(t) microlenses 510 that formthe image in the vertical axis and the image is assumed to have anaspect ratio such that the microlens array 508 is scanned to image N_(t)points in the horizontal axis, then the dimensions of the area to beilluminated on the microlens array are approximately D_(x)=N_(t)D₅ andD_(y)=N_(t)D₅. In the case that the multimode fiber 502 has a core ofcircular cross section, the minimum magnification M_(min) can be writtenapproximately as:

$\begin{matrix}{M_{\min} \cong {\sqrt{2}{\frac{N_{t}D_{5}}{D_{F}}.}}} & (11)\end{matrix}$

The square root of 2 factor is needed so that the entire image diagonalis filled. If the multimode fiber 502 has a core of square cross sectionof dimension D_(F), the minimum magnification M_(min) is writtenapproximately as:

$\begin{matrix}{M_{\min} \cong {\frac{N_{t}D_{5}}{D_{F}}.}} & (12)\end{matrix}$

If the multimode fiber 502 has a core of rectangular cross section withdimensions height D_(FH) and width D_(FW), the minimum magnificationM_(min) is written approximately as:

$\begin{matrix}{M_{\min} \cong {{\max\left( {\frac{N_{t}D_{5}}{D_{FW}},\frac{N_{t}D_{5}}{D_{FH}}} \right)}.}} & (13)\end{matrix}$

If the multimode fiber 502 has a core of circular cross section, Eqns.7, 8 and 11 may be used with the paraxial approximation to derive thefollowing relationship between the product of the core diameter D_(F) ofthe multimode fiber 502 and the exit angle θ₁:D _(F)θ₁≦√{square root over (2)}N _(t) D _(P) NA _(MS).  (14)

If the multimode fiber 502 has a core of square cross section, Eqns. 7,8, and 12 may be used with the paraxial approximation to derive ananalogous relationship to that of Eqn. 14. In this case, the square rootof two factor is not present, such that:D _(F)θ₁ ≦N _(t) D _(P) NA _(MS).  (15)

In the case that the core of the multimode fiber 502 has a rectangularcross section, the square dimension D_(F) in Eqn. 15 is replaced by therectangular height D_(FH) or rectangular width D_(FW) that gives thelargest value of N_(t)/D_(FW) or N_(t)/D_(FH).

Assuming light from the multimode fiber 502 exits into air (refractiveindex n=1), according to Eqn. 1, the numerical aperture NA_(F) isrelated to the exit angle θ₁ of the multimode fiber 502 byNA_(F)=sin(θ₁). For small angles θ₁, sin(θ₁) is approximately equal toθ₁, that is sin(θ₁)≅θ₁. Therefore, the numerical aperture NA_(F) of thefiber is approximately equal to the angle θ₁, that is NA_(F)≅θ₁.Therefore, for the case in which the multimode fiber 502 has a core ofcircular cross section, the product of the core diameter D_(F) and thenumerical aperture NA_(F) of the multimode fiber 502 should be limitedto the value on the right-hand side of Eqn. 14 in order to achieveefficient illumination of a sample. Similarly, for the case in which themultimode fiber 502 has a core of square or rectangular cross section,the product of the dimension D_(F) (or D_(FH) or D_(FW)) and thenumerical aperture NA_(F) of the multimode fiber 502 should be limitedto the value on the right-hand side of Eqn. 15 in order to achieveefficient illumination of a sample. Larger values for the product of thecore dimension D_(F) and the numerical aperture NA_(F) may be used butmay result in a reduction in the transmission of light from themultimode fiber 502 through the pinholes 514 or a reduction in the lightavailable to the detector. However, it should be noted that a multimodefiber of larger core dimensions than would be predicted by Eqn. 14 orEqn. 15 could be used if the radiation coupled into the incident end(not shown) of the multimode fiber had an input numerical aperture ofless than the numerical aperture NA_(F) of the multimode fiber. In thiscase, the exit angle θ₁ of light emitted from the multimode fiber mightbe less than that determined by the numerical aperture NA_(F) of themultimode fiber, depending on the physical properties associated withthe composition and configuration of the multimode fiber.

The relation given by Eqn. 14 (for a fiber having a core of circularcross section) or Eqn. 15 (for a fiber having a core of square orrectangular cross section) may be used to select an appropriatemultimode fiber for efficient illumination of a sample to be probed by amulti-focal confocal microscope system. For example, given a multimodefiber 502 having a particular core diameter D_(F), and lenses 504 and506 that produce a particular minimum magnification M_(min), therelationship of Eqn. 14 may be used to select the appropriate numericalaperture NA_(F) of the multimode fiber 502 for efficient illumination ofthe sample to be probed by the microscope, where the microscope has anacceptance numerical aperture NA_(MS), with the microlens array 508 andthe pinhole array 512 configured as described with respect to FIGS. 5and 6.

In the case of a multi-focal confocal subsystem where the pinholes 514are centered at the focal points of the corresponding microlenses 510,improved efficiency may be achieved if the light-coupling optics areconfigured so that the light rays that are parallel to the optical axis518 at the plane of the distal end 500 of the multimode fiber 502 aremaintained parallel to the optical axis 518 at the image plane by theimaging optics. However, in the case of a multi-focal confocal subsystemwhere the pinholes 514 are offset from the focal points of theircorresponding microlenses 510, improved efficiency may be achieved ifthe light-coupling optics are configured so that rays parallel to theoptical axis 518 at the distal end 500 of the multimode fiber 502 stillpass through the centers of the pinholes 514.

The relationships expressed in Eqns. 14 and 15 are related to an etendueΩ of the optical system. Etendue is a measure of the potentialthroughput of light in an optical system. An etendue of light emittedfrom a fiber can be defined to be the product of the solid anglesubtended by the emission and the area of the distal end of the fiber.In a single point scanning confocal system using a single mode fiber102, as described with respect to FIG. 1, the relationship between thecore diameter D_(F) and divergence angle θ₁ from the single mode fiber102 is fixed by diffraction theory. The size D_(P) of the pinholes 108is also typically chosen to be at or near the diffraction limited spotsize for light converging at the acceptance numerical aperture NA_(MS)of the microscope. A smaller pinhole 108 will reduce the lighttransmission to the sample and a larger pinhole 108 will reduce theresolution. The spot sizes of the light exiting the single mode fiber102 and the light at the pinhole 108 are both determined by diffractiontheory and have the relationship:D _(F)θ₁ =D _(P)θ₂  (16)

An etendue of the single point scanning confocal system is proportionalto the square of the left hand side of Eqn. 16, that is (D_(F)θ₁)²,where the square of core dimension D_(F) is proportional to the exitmode area of the fiber and, for small exit angles θ₁, the square of theexit angle θ₁ is proportional to the solid angle of light exiting thefiber. An etendue of the light exiting the fiber is thus fixed bydiffraction theory and the fiber is limited to being a diffractionlimited source, thus requiring it to be a single mode fiber.

As in the case of the single point scanning confocal system, for amulti-focal confocal system, the square of the left hand side of Eqn. 14or Eqn. 15, that is (D_(F)θ₁)², is proportional to an etendue of lightexiting the fiber. It is apparent from the squares of the right-handsides of Eqns. 14 and 15 that, in general, when using fiber to deliverradiation in a multi-focal confocal system, the etendue of light emittedfrom the distal end of the fiber and imaged onto the microlens array isproportional to the total number of pinholes to be illuminated. This canbe compared to a single point scanner where the etendue of the lightsource is fixed, as shown by Eqn. 16. The increase in etendue due to themultiple pinholes in a multi-focal confocal system with a microlensarray allows for the use of a light source having a larger etendue, suchas a multimode fiber, while still maintaining good light throughputthrough the optical system.

In a multi-focal confocal subsystem illustrated in FIGS. 5 and 6, if thefocal length F₅ of the microlenses 510 is designed to optimally transmitlight from a single mode fiber through the pinholes 514, the maximumangle of light rays passing through a particular pinhole 514 will beslightly larger if the single mode fiber is replaced with the multimodefiber 502. Therefore, the use of the multimode fiber 502 may cause someextra light loss through the microscope optics, however this increase inangle is generally small and may result in very little additional lightbeing lost. The small size of this angle may be demonstrated byconsidering a light ray 616 that passes through the edge of themicrolens 602 and proximal the edge of the pinhole corresponding to themicrolens 602, as illustrated in FIG. 6-2. Relative to the optical axis518, the light ray 616 has the largest angle through the pinhole. Thelight ray 616 passes through the pinhole at an angle ofθ₃=(D₅+D_(P)(2F₅). As previously described with respect to Eqn. 8, thefocal length F₅ and diameter D₅ of the microlenses 510 are typicallydesigned so that collimated light incident on the microlenses 510 willconverge at an angle to match the acceptance numerical aperture NA_(MS)of the microscope. A rearrangement of Eqn. 8 indicates that the focallength F₅ and diameter D₅ may be designed such that a ratio of thediameter D₅ to the focal length F₅ is approximately equal to twice theacceptance numerical aperture NA_(MS) of the microscope, that isD₅/F₅≅=2NA_(MS). By design, the diameter D_(P) of the pinhole is muchsmaller than the diameter D₅ of the microlens 602. This means that thelight angle of divergence of light into the pinhole will still closelymatch the acceptance numerical aperture NA_(MS) of the microscope.

The lens configurations described with respect to FIGS. 5, 6-1 and 6-2are merely examples. It is contemplated that any optical system may beused that images the distal end of a multimode fiber onto, orsufficiently close to, the plane of the microlens array to achievesubstantially uniform illumination at that plane. Furthermore, theproposed technology presented herein should not be considered limited tomulti-focal confocal microscopes. For example, in the absence of amicrolens array (i.e., in multiplexed confocal microscopy), the distalend of a multimode fiber may be imaged directly onto a pinhole array. Asis typical in multiplexed confocal microscopy, without the microlensarray, much of the light from the multimode fiber will not pass throughthe pinholes. Therefore, maintaining light rays parallel to the opticalaxis of the multimode fiber may be of little or no benefit inmultiplexed confocal microscopy. However, the uniform illumination ofthe pinholes, and the resulting uniform illumination of the sample afterthe complete scan, will remain. Even in the absence of microlenses, theuniform illumination allows for some improvement in light utilizationefficiency since there is no need to restrict the portion of the beamused for illumination. Similarly, in the absence of both microlenses andpinholes, it is contemplated that any optical system may be used thatimages the distal end of a multimode fiber onto, or sufficiently closeto, the plane of the sample to achieve substantially uniformillumination at that plane.

In Operation:

Operation of the multi-focal confocal microscope system 300 illustratedin FIG. 3 will now be described for the case that a core of themultimode fiber 208 has a circular cross section.

The radiation sources 218 generate radiation of different wavelengths inUV-visible-NIR spectral range. Optics of the light control andconditioning subunits 224 provide the radiation beam being guided intothe incident end 220 of the multimode fiber 208 by the lens 222 with apreferably circular cross section of a predetermined diameter. In thecase that the radiation sources 218 generate radiation beams of acircular cross section, the light control and conditioning subunits 224may expand the light from the radiation sources 218, providing it withthe predetermined diameter. In the case that the radiation sources 218generate light beams of an elliptical cross section, the light controland conditioning subunits 224 may compensate the beam ellipticity andoptimize beam diameter.

The folding mirror 232 and the dichroic mirrors 234 couple the laserbeams of the predetermined diameter, providing a single multi-wavelengthcollimated beam of exciting radiation incident on the lens 222.

The lens 222 focuses the exciting radiation emitted by the radiationsources 218 onto the incident end 220 of the multimode fiber 208. Theexciting radiation is guided from the incident end 220 through themultimode fiber 208 to the distal end 230. As the light is being guidedthrough the multimode fiber 208, the vibrating mechanical driver 308generates vibrations in a section of the multimode fiber 208, resultingin fast variations of the optical path lengths of individual rays in themultimode fiber 208, thereby randomizing the phase of light as it isguided through the multimode fiber 208.

The exciting radiation is emitted from the distal end 230 of themultimode fiber 208 and is turned into diverging rays with a maximumspread-angle dependent on the numerical aperture NA_(F) of the multimodefiber 208. The light is then received by the light-coupling unit 236,where it passes through the lenses 242 and 244. The lenses 242 and 244and the multimode fiber 208 have been configured to image the distal end230 of the multimode fiber 208 onto, or sufficiently close to, the plane352 of the microlens array disk 310 such that the radiant intensity oflight at the plane 352 of the microlens array disk 310 is substantiallyuniform. Specifically, since the lenses 242 and 244 have been selectedto provide a magnification equal to or greater than the minimummagnification M_(min) according to Eqn. 11, the size of the image of thedistal end 230 of the multimode fiber 208 at the plane 352 of themicrolens array disk 310 is sufficient to illuminate with substantialuniformity all the pinholes 316 that are used by the detector 258 toconstruct the confocal image of the sample 248.

The imaged radiation at the microlens array disk 310 is collected by themicrolenses 312 and focused onto the corresponding pinholes 316 in thepinhole array disk 314 after passing through dichroic mirror 254. Theradiation exiting the pinhole array disk 314 is converged to the sample248 by the objective lens 246.

Assuming the lenses 504 and 506 are examples of the lenses 242 and 244respectively, and assuming that the microlens array 508 and the pinholearray 512 are examples of the microlens array disk 310 and the pinholearray disk 314, respectively, the paths of light rays emitted from thedistal end 230 of the multimode fiber 208 and passing through thepinholes 316 of the pinhole array disk 314 may be similar to the examplepaths of the light rays illustrated in FIGS. 5 and 6.

Since the core diameter D_(F) and the numerical aperture NA_(F) of themultimode fiber 208 have been selected to approximately follow therelation of Eqn. 14, the etendue of light emitted from the distal end230 of the multimode fiber 208 does not substantially exceed the etendueof light passing through the pinholes 316 of the pinhole array 314,thereby achieving efficient illumination of the sample 248.

A signal from the sample 248, for example a fluorescence signal, passesback through the objective lens 246, and is converged to the individualpinholes 316 of the pinhole array disk 314. The fluorescence signalpassing through the individual pinholes 316 is reflected by the dichroicmirror 254, so as to be imaged onto a sensor (not shown) of the detector258 via the relay lens 260.

The plane of the pinhole array disk 314, the sample plane 250, and aplane of an image sensor (not shown) of the detector 258 are arranged tobe conjugate with each other optically. The pinhole array disk 314 isrotated jointly with the microlens array disk 310 at a constant speed,and a converged light spot on the sample 248 is scanned with thepinholes 316 moved by the rotation. An optical sectional image, that isa confocal image, of the sample 248 is imaged onto the image sensor ofthe detector 258. After a complete scan in which the pinholes 316 arerotated, the sample 248 is uniformly illuminated.

The examples described with respect to FIGS. 2 and 3 include threeradiation sources and three light control and conditioning subunits.Alternatively, it is possible to include one or more radiation sourcesand provide as many different wavelengths of the exciting radiation. Itis also possible to improve the signal-to-noise ratio of the images if aradiation source with only one of the wavelengths is selected and putinto use by the switching means and fluorescent light is received in atime division manner.

Example Calculations for Circular Core Multimode Fiber

Example calculations of the optics of the multi-focal confocalmicroscope system 300 with the multimode fiber 208 having a core ofcircular cross section will now be presented. The following calculationswill be done for the multimode fiber 208 having a core diameter of 200μm. This is a common core diameter that is commercially available.

In this example, the properties of the multi-focal confocal microscopesystem 300 are as follows: the diameter of D₅ of the microlenses 312 isequal to 0.25 mm; the focal length F₅ of the microlenses 312 is equal to10 mm; the diameter D_(P) of the pinholes 316 is equal to 50 μm; and theacceptance numerical aperture NA_(MS) of the microscope 238 is equal to0.0125 (where the numerical aperture of the microlenses 312 is designedto match that of the microscope 238). The detector 258 and themicroscope 238 used in this example dictate that the illuminated area onthe microlenses 312 should be at least D_(x) by D_(y) whereD_(x)=D_(y)=10 mm.

Given the diameter D₅ of the microlenses 312 and the dimension D_(x),the total number N_(t) of microlenses 312 to be illuminated along onedimension is N_(t)=D_(x)/D₅≅10 mm/0.25 mm=40. Assuming the maximum exitangle θ₁ is substantially equal to the numerical aperture NA_(F) of themultimode fiber 208, that is θ₁≅NA_(F), Eqn. 14 dictates that thenumerical aperture NA_(F) of the multimode fiber 208 should notsubstantially exceed NA_(F)≅√{square root over(2)}*N_(t)*NA_(MS)*D_(P)/D_(F)≅1.41*40*0.0125*50 μm/200 μm≅0.176.

A numerical aperture NA_(F) of 0.176 may be achieved either by thedesign of the fiber core-cladding index differential or by coupling intoa larger numerical aperture fiber at a smaller numerical aperture thanthe design numerical aperture. In practice, it may be desired to use aslightly smaller numerical aperture than that calculated using Eqn. 14in order to account for alignment errors and deviations from the idealassumptions used to derive Eqn. 14. A reasonable value might beNA_(F)=0.15.

Although this example uses a multimode fiber with a core diameter of 200μm, it is contemplated that a smaller or larger core diameter which istechnically reasonable may be used if the limiting condition of Eqn. 14is approximately satisfied.

With the multimode fiber 208 having a core diameter D_(F) equal to 200μm, the minimum magnification M_(min) of the lenses 242 and 244 iscalculated according to Eqn. 11 as M_(min)≅√{square root over(2)}*N_(t)*D₅/D_(F)≅(1.41*10 mm/0.2 mm)≅70.5. This minimum magnificationM_(min) will exactly illuminate the diagonal of the sample 248 such thatthe image of the sample 248 on the detector 258 fills the active regionof the detector 258. In practice, a magnification M that is slightlylarger than the minimum magnification M_(min), for example M=75, mightbe used to ensure that all of the pinholes used to construct theconfocal image are illuminated in the presence of any smallmisalignments of the optics.

Given the lenses 242 and 244 with a magnification M equal to 75 and themultimode fiber 208 with a numerical aperture NA_(F) equal to 0.15, itis possible to confirm that exciting radiation will pass efficientlythrough the pinholes 316. According to Eqn. 7, the diameter of thegeometric spot size at the pinholes 316 is equal to twice the product ofthe numerical aperture at the microlenses 312 (NA_(F)/M) and the focallength F₅ of the microlenses 312, that is 2*(NA_(F)/M)*F₅=2*(0.15/75)*10mm=40 μm. Given that the diameter D_(P) of the pinholes 316 is 50 μm, itfollows that the exciting radiation will pass efficiently through thepinholes 316.

In general, it is desired to keep the focal lengths F₃ and F₄ of thelenses 242 and 244 as small as possible to reduce the size of theoptical system. For example, the lens 242 may be chosen to have a focallength F₃ of 3 mm since this value is not too difficult to manufacture.According to Eqn. 4, it is desired that the ratio of the focal length F₃of the lens 242 over the focal length F₄ of the lens 244 is equal to themagnification M. Therefore, the focal length F₄ of the lens 244 iscalculated as F₄=M*F₃=75*3 mm=225 mm. It follows that the lens 242should be placed a distance U₁=F₃=3 mm from the distal end 230 of themultimode fiber 208; the lens pair 242 and 244 should be separated by adistance U₃=F₃+F₄=3 mm+225 mm=228 mm; and the microlens array disk 310should be placed a distance U₂=F₄=225 mm from the lens 244.

This example was initiated using a given core diameter D_(F) of themultimode fiber 208 equal to 200 μm. As discussed previously withrespect to Eqn. 14, it is the product of the core diameter D_(F) and thenumerical aperture NA_(F) of the multimode fiber 208 that is limited bythe right-hand side of Eqn. 14. Therefore, it would have been just asvalid to initiate the example calculations using a given numericalaperture NA_(F) and then calculating the desired core diameter D_(F).

It should be noted that the core diameter D_(F) of a specific fiber isalways fixed. However, it is possible to obtain a smaller effectivenumerical aperture NA_(F) as compared to the actual numerical apertureNA_(F). This can be done, for example, by under filling the inputnumerical aperture of the multimode fiber when coupling light into themultimode fiber. Accordingly, there is a small amount of room to adjustthe numerical aperture NA_(F) as seen by the multi-focal confocalmicroscope.

Example Calculations for Square or Rectangular Core Multimode Fiber

Example calculations of the optics of the multi-focal confocalmicroscope system 300 with the multimode fiber 208 having a core ofsquare or rectangular cross section will now be presented. The followingcalculations will be done for the multimode fiber 208 having a squarecore of side length 125 μm. The remaining parameters are the same asthose used in the previous example for the circular core fiber,including the diameter D₅ of the microlenses 312 equal to 0.25 mm, thefocal length F₅ of the microlenses 312 equal to 10 mm, the diameterD_(P) of the pinholes 316 equal to 50 μm, the acceptance numericalaperture NA_(MS) of the microscope 238 equal to 0.0125, and thedimensions of the area to be illuminated on the microlens array 310equal to D_(x) by D_(y), where D_(x)=D_(y)=10 mm. As in the previousexample, the total number N_(t) of microlenses 312 or pinholes 316 alongone dimension is N_(t)=D_(x)/D₅=10 mm/0.25 mm=40.

In this case, Eqn. 15 is used to determine that the numerical apertureNA_(F) of the multimode fiber 208 should not substantially exceedNA_(F)=N_(t)*NA_(MS)*D_(P)/D_(F)≅40*0.0125*50 μm/125 μm≅=0.2. As in theprevious example, it may be desired to use a slightly smaller numericalaperture than that calculated using Eqn. 15 in order to account foralignment errors and deviations from the ideal assumptions used toderive Eqn. 15. A reasonable value might be NA_(F)=0.18.

The minimum magnification M_(min) should be a ratio of the desired imagesize to the relevant dimension of the multimode fiber 208. According toEqn. 12, for a multimode fiber with a square or rectangular core, theminimum magnification M_(min) is calculated asM_(min)=N_(t)*D₅/D_(F)=(10 mm/0.125 mm)=80. As with the circular corefiber, the magnification M may be made a somewhat larger than theminimum magnification M_(min) to provide some tolerance to alignmenterrors. A magnification M=85 is a reasonable choice.

Given the lenses 242 and 244 with a magnification M equal to 85 and themultimode fiber 208 with a numerical aperture NA_(F) equal to 0.18, itis possible to confirm that exciting radiation will pass efficientlythrough the pinholes 316. According to Eqn. 7, the diameter of thegeometric spot size at the pinholes 316 is equal to twice the product ofthe numerical aperture at the microlenses 312 (NA_(F)/M) and the focallength F₅ of the microlenses 312, that is 2*(NA_(F)/M)*F₅=2*(0.18/85)*10mm=42 μm. This confirms that the exciting radiation will passefficiently through the 50-μm pinholes 316.

If the focal length F₃ of the lens 242 is chosen to be 3 mm, the focallength F₄ of the lens 244 is calculated according to Eqn. 4 asF₄=M*F₃=85*3 mm=255 mm. It follows that the lens 242 should be placed adistance U₁=F₃=3 mm from the distal end 230 of the multimode fiber 208;the lenses 242 and 244 should be separated by a distance U₃=F₃+F₄=3mm+255 mm=258 mm; and the lens 244 should be placed a distance U₄=F₄=255mm from the microlens array disk 310.

The technique of imaging a distal end of a multimode fiber to providesubstantially uniform illumination is not limited to multiplexedconfocal microscopy using a microlens array. The techniques described toproduce uniform illumination may be used in multiplexed confocalmicroscopy where a pinhole array is used without a microlens array, orin any microscope system that benefits from uniform, wide-areaillumination. Microscopy techniques that can be used include, but arenot limited to, wide-field imaging, fluorescence recovery afterphoto-bleaching (FRAP), fluorescence lifetime imaging (FLIM), structuredillumination (SIM), photo-activated localization microscopy (PALM) andstochastic optical reconstruction microscopy (STORM).

In wide-field and bright field microscopy the image of a distal end of amultimode fiber may be imaged onto a sample of a microscope.Intermediate optics between the distal end of the multimode fiber andthe objective lens of the microscope may be used such that the distalend of the multimode fiber is imaged onto the sample. Alternatively, thedistal end of the multimode fiber may be imaged to one or more planesconjugate to the sample plane. In the example of FIG. 2, the lenses 242and 244 are used to image the distal end 230 of the multimode fiber 208to the conjugate plane 252. The uniform illumination at the conjugateplane 252 is imaged by the microscope onto the sample 248, thusproviding substantially uniform illumination of the sample 248. In PALMor STORM methods, the distal end 230 of the multimode fiber 208 isimaged onto the sample 248 in the same way as wide-field or bright fieldmicroscopy. An alternate method of wide-field microscopy may be achievedin the configuration of FIG. 3 with the pinhole apertures 316 being madevery large or by the removal of the pinhole array disk 314 altogether.

In a FRAP system, a distal end of a multimode fiber may be imaged onto aplane that is conjugate to the plane of a sample. Uniform illuminationis then present at the conjugate plane. A section of the substantiallyuniform illumination is selected by the FRAP system. The sectionselected corresponds to the sections of the sample that will bephotobleached. Selection may be achieved with a digital micromirrordevice, physical aperture or other techniques known to those skilled inthe art. The selected sections in the conjugate plane are transferred byan objective lens to the sample with uniform illumination across theselected section.

The FLIM technique measures the fluorescence lifetime at a specificpoint in a sample. In some implementations of FLIM, the sample isilluminated in an area sufficient to substantially illuminate the areaof the microscope image. The incoming illumination is time modulatedwith pulsed, sinusoidal or otherwise time selective modulation. Imaginga distal end of a multimode fiber onto the sample or to a planeconjugate to the sample plane may provide substantially uniformillumination of the sample. Time modulation may be applied to theilluminating light either before coupling to the multimode fiber orafter exiting the multimode fiber. Modulating methods that may be usedinclude, but are not limited to, directly modulating light emitted fromthe radiation source or plurality of radiation sources, placing amodulation means between the radiation source or plurality of sourcesand the multimode fiber, or by placing a modulation means after thedistal end of the multimode fiber.

Structured illumination, or patterned illumination, or 3D structuredillumination microscopy (3D-SIM), applies a pattern or structure to themicroscope illumination. Multiple images with different patterns areexposed separately and an image processing means is used to reconstructan image that can exceed the diffraction limit of a traditionalmicroscope. More advanced patterning techniques can be used to recoversectioned images of the sample. Imaging a distal end of a multimodefiber onto a plane conjugate to the sample plane may produce uniformillumination for the structured illumination. The uniform illuminationat the conjugate image plane is patterned with various techniques knownto those skilled in the art and the subsequently patterned illuminationis imaged onto the sample.

While certain features of the technology have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes.

What is claimed is:
 1. A method comprising: imaging a distal end of amultimode fiber (i) onto a plane, or (ii) sufficiently close to theplane such that a radiant intensity of light at the plane issubstantially uniform, wherein a transverse distribution of lightexiting the distal end is substantially uniform and the light exitingthe distal end passes through a light-coupling unit before reaching theplane, wherein the light-coupling unit and a segmented focusing array offocusing elements at the plane are comprised in a multi-focal confocalsubsystem, and wherein light passing through the focusing elements isfocused at or substantially near a plane that is conjugate to a sampleplane at which a sample is to be probed by a microscope, the microscopecomprising at least an objective.
 2. The method as claimed in claim 1,wherein the multimode fiber is a step-index multimode fiber.
 3. Themethod as claimed in claim 1, wherein the light-coupling unit provides amagnification such that an area of substantially uniform illumination atthe sample plane is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of a detector.
 4. Themethod as claimed in claim 1, wherein a core of the multimode fiber isof circular cross section.
 5. The method as claimed in claim 1, whereina core of the multimode fiber is of rectangular cross section.
 6. Themethod as claimed in claim 1, wherein a core of the multimode fiber isof square cross section.
 7. The method as claimed in claim 1, whereinthe multi-focal confocal subsystem further comprises an aperture arrayof apertures that is positioned at the plane that is conjugate to thesample plane.
 8. The method as claimed in claim 7, wherein the multimodefiber and the multi-focal confocal subsystem are configured such thatlight exiting the distal end of the multimode fiber parallel to anoptical axis of the multimode fiber passes through centers of one ormore of the apertures.
 9. A multi-focal confocal subsystem comprising: alight-coupling unit to be optically coupled to a multimode fiber; asegmented focusing array of focusing elements at a segmented focusingarray plane; and an aperture array of apertures, the multi-focalconfocal subsystem (i) configured such that light exiting a distal endof the multimode fiber and passing through the light-coupling unit isimaged onto the segmented focusing array plane, or (ii) configured suchthat light exiting the distal end and passing through the light-couplingunit is imaged sufficiently close to the segmented focusing array planesuch that a radiant intensity of the light at the segmented focusingarray plane is substantially uniform, wherein a transverse distributionof light exiting the distal end is substantially uniform, and the lightexiting the distal end passes through the light-coupling unit andreaches the segmented focusing array plane without passing through theaperture array.
 10. The multi-focal confocal subsystem as claimed inclaim 9, configured such that light exiting the distal end of themultimode fiber parallel to an optical axis of the multimode fiberpasses through centers of one or more of the apertures.
 11. Amulti-focal confocal microscope system comprising: a multi-focalconfocal subsystem, comprising: a light-coupling unit to be opticallycoupled to a multimode fiber; a segmented focusing array of focusingelements at a segmented focusing array plane; and an aperture array ofapertures, and a microscope comprising at least an objective, themulti-focal confocal subsystem (i) configured such that light exiting adistal end of the multimode fiber and passing through the light-couplingunit is imaged onto the segmented focusing array plane, or (ii)configured such that light exiting the distal end and passing throughthe light-coupling unit images the distal end sufficiently close to thesegmented focusing array plane such that a radiant intensity of thelight at the segmented focusing array plane is substantially uniform,wherein a transverse distribution of light exiting the distal end issubstantially uniform, wherein the aperture array is positioned betweenthe segmented focusing array plane and a sample plane, wherein lightexiting the apertures is imaged onto, or sufficiently close to, thesample plane such that a radiant intensity of the light at the sampleplane is substantially uniform after a complete scan, and wherein thesample plane is a plane at which a sample is to be probed by themicroscope.
 12. The multi-focal confocal microscope system as claimed inclaim 11, further comprising the multimode fiber.
 13. The multi-focalconfocal microscope system as claimed in claim 11, further comprisingthe multimode fiber and wherein the multimode fiber is a step-indexmultimode fiber.
 14. The multi-focal confocal microscope system asclaimed in claim 11, further comprising the multimode fiber and whereina core of the multimode fiber is of circular cross section.
 15. Themulti-focal confocal microscope system as claimed in claim 11, furthercomprising the multimode fiber and wherein a core of the multimode fiberis of rectangular cross section.
 16. The multi-focal confocal microscopesystem as claimed in claim 11, further comprising the multimode fiberand wherein a core of the multimode fiber is of square cross section.17. The multi-focal confocal microscope system as claimed in claim 11,further comprising the multimode fiber, wherein a core of the multimodefiber is of circular cross section, the light-coupling unit provides amagnification such that an area of substantially uniform illumination atthe sample plane is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of a detector, and afirst value does not substantially exceed a second value, wherein thefirst value is a product of a numerical aperture of the multimode fiberand of a diameter (D_(F)) of the core, and the second value is a productof a number (√{square root over (2)}N_(t)) of focusing elements along adiagonal of the imaged area, a length (D_(P)) of one of the apertures,and an acceptance numerical aperture (NA_(MS)) of the microscope. 18.The multi-focal confocal microscope system as claimed in claim 11,further comprising the multimode fiber, wherein a core of the multimodefiber is of square cross section, the light-coupling unit provides amagnification such that an area of substantially uniform illumination atthe sample plane is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of a detector, and afirst value not substantially exceed a second value, wherein the firstvalue is a product of a numerical aperture of the multimode fiber and ofa square dimension (D_(F)) of the core, and the second value is aproduct of a number (N_(t)) of focusing elements along a side of theimaged area, a length (D_(P)) of one of the apertures, and an acceptancenumerical aperture (NA_(MS)) of the microscope.
 19. The multi-focalconfocal microscope system as claimed in claim 11, further comprisingthe detector.
 20. The multi-focal confocal microscope system as claimedin claim 11, configured such that light exiting the distal end of themultimode fiber parallel to an optical axis of the multimode fiberpasses through centers of one or more of the apertures.
 21. Themulti-focal confocal microscope system as claimed in claim 11, furthercomprising the multimode fiber, wherein a core of the multimode fiber isof rectangular cross section, the light-coupling unit provides amagnification such that an area of substantially uniform illumination atthe sample plane is not substantially bigger than an imaged area of thesample plane that is imaged by any active region of a detector, and afirst value does not substantially exceed a second value, wherein thefirst value is a product of a numerical aperture of the multimode fiberand either a width (D_(FW)) or a height (D_(FH)) of the core, whicheveris smaller, and the second value is a product of a number (N_(t)) offocusing elements along a side of the imaged area, a length (D_(P)) ofone of the apertures, and an acceptance numerical aperture (NA_(MS)) ofthe microscope.